MRPL17 Antibody

What is MRPL17 and what cellular functions does it perform?

MRPL17 (mitochondrial ribosomal protein L17) is a protein component of the mitochondrial ribosome that plays a crucial role in mitochondrial protein synthesis . It is encoded by the MRPL17 gene (Gene ID: 63875) and has several synonyms including LIP2, L17mt, RPL17L, RPML26, MRP-L17, and MRP-L26 . The protein has a calculated molecular weight of approximately 20 kDa, though it is often observed at around 25 kDa in experimental conditions . MRPL17 is primarily localized in the mitochondrion and contributes to the functioning of the mitochondrial translation machinery, which is essential for the synthesis of proteins encoded by mitochondrial DNA, particularly those involved in oxidative phosphorylation .

What is the structure and key characteristics of commercially available MRPL17 antibodies?

MRPL17 antibodies are typically available as polyclonal antibodies raised in rabbits (rabbit IgG) . These antibodies are produced using immunogens such as recombinant fusion proteins containing sequences from human MRPL17. For example, one commercially available antibody (CAB15603) uses a recombinant fusion protein containing amino acids 86-175 of human MRPL17 (NP_071344.1) as the immunogen . Another antibody (17214-1-AP) is produced using the MRPL17 fusion protein Ag10997 . These antibodies are typically supplied in liquid form with storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and should be stored at -20°C .

What are the appropriate applications for MRPL17 antibodies in research?

MRPL17 antibodies are primarily used in Western blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . For Western blotting, the recommended dilution ranges from 1:500 to 1:2000 . These antibodies show reactivity with human, mouse, and rat samples, making them suitable for comparative studies across these species . Positive control samples that have been validated for use with these antibodies include 293T and MCF7 cell lines, as well as mouse kidney tissue . These antibodies are valuable tools for researchers investigating mitochondrial function, protein synthesis, and the role of MRPL17 in various disease contexts, particularly in cancer research .

How has MRPL17 been implicated in cancer pathogenesis, particularly in liver hepatocellular carcinoma?

Recent single-cell analysis studies have identified MRPL17 as one of 14 essential stem cell-related genes with significant prognostic value in liver hepatocellular carcinoma (LIHC) . Machine learning algorithms, particularly XGBOOST, have indicated that MRPL17 is strongly associated with patient prognosis and responses to immunotherapy in LIHC . Experimental validation has demonstrated that MRPL17 is upregulated in LIHC tissue samples and correlates with poor prognosis . Furthermore, MRPL17 expression positively correlates with KI67, a marker of cell proliferation . Functional analyses have connected MRPL17 to cell proliferation, epithelial-mesenchymal transition (EMT), and oxidative phosphorylation in LIHC . Similar findings have been reported in other cancer types, with MRPL17 identified as a prognostic marker in lung cancer and endometrial cancer through bioinformatics analyses .

What is the relationship between MRPL17 expression and immune cell infiltration in tumor microenvironments?

Analysis of TCGA-LIHC dataset samples sorted by MRPL17 expression levels has revealed significant differences in immune cell infiltration patterns . Specifically, variations were observed in the quantities of activated myeloid dendritic cells, M1 and M2 macrophages, granulocyte-monocyte progenitors, hematopoietic stem cells, endothelial cells, regulatory T cells (Tregs), mast cells, CD4+ Th2 T cells, and B cells . The expression of MRPL17 has been correlated with the fractions of immune cell infiltration using algorithms such as XCELL and TIP . Additionally, significant expression differences in six immune checkpoint-related genes were discovered between high and low MRPL17 expression groups . The TIDE algorithm revealed that patients with high MRPL17 expression exhibited elevated TIDE scores, suggesting less effective responses to immunotherapy . These findings highlight the potential role of MRPL17 in modulating the tumor immune microenvironment and affecting immunotherapy outcomes.

What bioinformatic approaches have been used to establish MRPL17 as a prognostic biomarker?

Multiple bioinformatic approaches have been employed to establish MRPL17 as a prognostic biomarker in LIHC . Single-cell analysis techniques were used to identify stem cell-associated genes and assess their prognostic significance for LIHC patients . Nonnegative matrix factorization (NMF) cluster analysis was employed to evaluate the efficacy of immunotherapy in relation to these genes . A diagnostic model for LIHC was developed and validated through multiple datasets using various machine learning clustering methods . The XGBOOST algorithm identified MRPL17 as the most significant prognostic gene among those associated with stem cells . The ssGSEA algorithm was used to assess enrichment scores for multiple pathways, revealing a positive relationship between MRPL17 expression and tumor proliferation as well as EMT . These computational approaches have collectively established MRPL17 as a promising prognostic biomarker with potential implications for treatment strategies.

What are the optimal protocols for immunofluorescence detection of MRPL17 in tissue samples?

For immunofluorescence detection of MRPL17 in tissue samples, the following protocol has been validated in LIHC tissue microarrays :

• Sample Preparation:

  • Deparaffinize paraffin slices by immersion in two tanks of xylene for 15 minutes each.

  • Rehydrate through a graded ethanol series (absolute ethanol, 95%, 85%, 75%) and distilled water, 5 minutes each.

• Antigen Retrieval:

  • Place sections in a repair box containing pH 9.0 EDTA alkaline antigen repair solution.

  • Heat in a pressure cooker for 2 minutes, then allow natural cooling.

  • Wash three times with PBS (pH 7.4) for 5 minutes each with shaking.

• Peroxidase Blocking:

  • Incubate in 3% hydrogen peroxide solution at room temperature in the dark for 15 minutes.

  • Apply blocking solution evenly and block at room temperature for 30 minutes.

• Primary Antibody Incubation:

  • Apply diluted MRPL17 antibody (bs-17773R) and incubate overnight at 4°C.

  • Wash three times with PBS for 5 minutes each.

• Secondary Antibody and Detection:

  • Apply poly-HRP secondary antibody corresponding to the primary antibody species.

  • Incubate at room temperature in the dark for 10-20 minutes.

  • Apply TSA fluorescent dye solution uniformly and incubate at room temperature for 15 minutes.

  • Apply ready-to-use DAPI dye and incubate in the dark at room temperature for 10 minutes.

• Imaging:

  • Mount slides and obtain images using a fluorescence microscope.

• Quantification:

  • Rate intensity of immunostaining on a scale from 0 to 3.

  • Measure percentage of positive staining on a scale from 1 to 4.

  • Calculate final expression score by multiplying intensity score by percentage scale score .

What are the recommended Western blotting conditions for optimal MRPL17 detection?

For optimal detection of MRPL17 using Western blotting, the following recommendations are based on validated antibody specifications:

For troubleshooting non-specific bands, it is recommended to optimize antibody concentration, blocking conditions, and washing steps. The observed molecular weight of 25 kDa versus the calculated 20 kDa may be due to post-translational modifications, which should be taken into consideration during result interpretation .

How can researchers validate MRPL17 antibody specificity for their experimental system?

To validate MRPL17 antibody specificity for a particular experimental system, researchers should consider implementing the following approaches:

• Positive and Negative Controls:

  • Use validated positive control samples such as 293T, MCF7 cell lines, or mouse kidney tissue .

  • Include negative controls such as tissues or cells known not to express MRPL17 or with MRPL17 knockdown/knockout.

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide (e.g., recombinant fusion protein containing amino acids 86-175 of human MRPL17) .

  • Compare staining patterns with and without peptide competition to identify specific signals.

• Multiple Antibody Validation:

  • Use multiple antibodies targeting different epitopes of MRPL17 to confirm specificity.

  • Compare staining patterns between different antibodies to identify consistent signals.

• Molecular Weight Verification:

  • Verify that the detected band appears at the expected molecular weight (approximately 25 kDa observed, 20 kDa calculated) .

  • Consider possible post-translational modifications that may affect migration on SDS-PAGE.

• Genetic Manipulation:

  • Perform siRNA knockdown or CRISPR/Cas9 knockout of MRPL17 to validate signal specificity.

  • Overexpress MRPL17 in a system with low endogenous expression to confirm signal enhancement.

• Cross-Reactivity Testing:

  • Test the antibody in multiple species if cross-reactivity is claimed (human, mouse, rat) .

  • Validate specificity in each species separately.

• Sequential Epitope Analysis:

  • Compare the immunogen sequence with other proteins to identify potential cross-reactive epitopes.

  • The sequence used for CAB15603 antibody is: IPKL FQVL APRY KDQT GGYT RMLQ IPNR SLDR AKMA VIEY KGNC LPPL PLPR RDSH LTLL NQLL QGLR QDLR QSQE ASNH SSHT AQTP GI .

How should researchers interpret discrepancies between MRPL17 expression data across different experimental platforms?

When encountering discrepancies in MRPL17 expression data across different experimental platforms, researchers should consider several factors:

• Methodological Differences:

  • Different antibodies may target different epitopes of MRPL17, potentially resulting in varying sensitivities and specificities .

  • The observed molecular weight of MRPL17 (25 kDa) differs from the calculated weight (20 kDa), which may lead to discrepancies when using techniques with different resolution capabilities .

• Sample Preparation Variations:

  • Different tissue or cell lysis methods may extract MRPL17 with varying efficiencies, especially given its mitochondrial localization.

  • Fixation methods for immunohistochemistry or immunofluorescence can affect epitope accessibility.

• Platform-Specific Considerations:

  • RNA-seq or qPCR data reflect mRNA levels, which may not directly correlate with protein levels detected by antibody-based methods.

  • Proteomic approaches may detect different post-translational modifications or protein complexes than antibody-based methods.

• Biological Variables:

  • MRPL17 expression varies across different tissues and cell types, with validated expression in 293T, MCF7 cell lines, and mouse kidney tissue .

  • Cancer tissues, particularly LIHC, show upregulated MRPL17 expression compared to normal tissues .

  • Expression may vary with disease progression, as MRPL17 has been associated with prognostic outcomes in LIHC .

• Validation Approaches:

  • Use multiple detection methods (e.g., Western blot, immunofluorescence, mass spectrometry) to verify expression patterns.

  • Include appropriate controls for each method and normalize data according to platform-specific standards.

  • Consider biological replicates to account for natural variation in expression levels.

What are the implications of MRPL17 co-expression with mitochondrial and stemness markers in cancer research?

The co-expression of MRPL17 with mitochondrial and stemness markers has several important implications for cancer research:

• Metabolic Reprogramming:

  • MRPL17, as a component of the mitochondrial ribosome, is involved in the synthesis of proteins required for oxidative phosphorylation .

  • Co-expression with other mitochondrial markers may indicate metabolic reprogramming in cancer cells, potentially reflecting a shift between glycolytic and oxidative metabolism.

  • This metabolic flexibility may contribute to cancer cell survival under varying nutrient conditions and stress.

• Cancer Stem Cell Properties:

  • MRPL17 has been identified as one of 14 essential stem cell-related genes in LIHC through single-cell analysis .

  • Co-expression with stemness markers suggests a potential role in maintaining cancer stem cell populations, which are implicated in tumor initiation, progression, and therapy resistance.

  • The positive correlation between MRPL17 and KI67 expression suggests an association with cell proliferation capabilities .

• Therapeutic Resistance Mechanisms:

  • Cancer stem cells are known to contribute to immune evasion within the tumor microenvironment .

  • MRPL17 expression has been associated with immune cell infiltration patterns and elevated TIDE scores, suggesting less effective responses to immunotherapy .

  • Targeting MRPL17 or its associated pathways may potentially enhance the efficacy of immunotherapeutic approaches.

• Prognostic Significance:

  • The co-expression pattern of MRPL17 with stemness markers has been linked to poor prognosis in LIHC patients .

  • Similar prognostic significance has been reported in other cancer types, including lung cancer and endometrial cancer .

  • This pattern may serve as a basis for developing combined biomarker panels for more accurate prognostication.

• Developmental Pathways:

  • Co-expression with stemness markers suggests involvement in developmental pathways that may be reactivated in cancer.

  • The association with epithelial-mesenchymal transition (EMT) indicates potential roles in cancer invasion and metastasis .

How can researchers integrate MRPL17 expression data with immune profiling to predict immunotherapy responses?

Researchers can integrate MRPL17 expression data with immune profiling to predict immunotherapy responses through several methodological approaches:

What are the knowledge gaps in understanding MRPL17's role in mitochondrial function and disease?

Despite recent advances in MRPL17 research, several significant knowledge gaps remain in understanding its role in mitochondrial function and disease:

• Structural-Functional Relationships:

  • Detailed structural analysis of MRPL17's interaction with other components of the mitochondrial ribosome is limited.

  • The specific role of MRPL17 in mitochondrial translation beyond its structural contribution to the ribosome remains poorly characterized.

  • How post-translational modifications of MRPL17 (suggested by the difference between calculated and observed molecular weights) affect function is unknown .

• Regulatory Mechanisms:

  • The transcriptional and post-transcriptional regulation of MRPL17 expression under normal physiological conditions is not well understood.

  • Factors that drive MRPL17 upregulation in cancer cells, particularly in LIHC, have not been fully elucidated .

  • The potential role of MRPL17 in mitochondrial stress responses and quality control mechanisms remains unexplored.

• Cancer Biology:

  • While MRPL17 has been identified as a prognostic marker in several cancers, the mechanistic basis for its contribution to cancer progression is not well defined .

  • How MRPL17 influences cancer stem cell properties and contributes to tumor heterogeneity requires further investigation .

  • The functional relationship between MRPL17 expression and immune cell infiltration in the tumor microenvironment needs mechanistic explanation .

• Therapeutic Targeting:

  • The feasibility and approaches for targeting MRPL17 as a therapeutic strategy in cancer have not been explored.

  • Whether inhibition of MRPL17 would specifically affect cancer cells without substantial toxicity to normal tissues remains unknown.

  • The potential for combining MRPL17-targeted therapies with immunotherapy approaches needs investigation .

• Systemic Effects:

  • The impact of MRPL17 expression on whole-organism metabolism and physiology has not been studied through knockout or transgenic animal models.

  • Potential roles of MRPL17 in aging and degenerative diseases associated with mitochondrial dysfunction remain unexplored.

What experimental approaches could help elucidate the potential therapeutic targeting of MRPL17 in cancer?

Several experimental approaches could help elucidate the potential therapeutic targeting of MRPL17 in cancer:

• Genetic Manipulation Studies:

  • CRISPR/Cas9 knockout or knockdown studies in cancer cell lines to assess effects on proliferation, migration, invasion, and stemness properties.

  • Conditional knockout models in animals to evaluate the impact of MRPL17 deletion on tumor initiation, progression, and metastasis in vivo.

  • Overexpression studies to determine if MRPL17 alone can drive oncogenic transformation or enhance malignant phenotypes.

• High-Throughput Screening:

  • Small molecule screening to identify compounds that can modulate MRPL17 expression or function.

  • PROTAC (Proteolysis Targeting Chimera) approaches to target MRPL17 for degradation.

  • Synthetic lethality screening to identify genes or pathways that, when inhibited, cause selective toxicity in cells with high MRPL17 expression.

• Structure-Based Drug Design:

  • Determination of the three-dimensional structure of MRPL17 and its interfaces with other mitochondrial ribosomal components.

  • In silico screening and rational design of compounds that can interfere with MRPL17's functional interactions.

  • Development of peptide-based inhibitors targeting specific domains of MRPL17.

• Combination Therapy Studies:

  • Evaluation of MRPL17 targeting in combination with immune checkpoint inhibitors, given its association with immune cell infiltration .

  • Testing combinations with conventional chemotherapeutics to assess potential synergistic effects.

  • Exploration of combinations with mitochondrial-targeted therapies to enhance cancer cell-specific metabolic vulnerabilities.

• Translational Research:

  • Development and validation of companion diagnostics for MRPL17 expression to identify patients most likely to benefit from targeted therapies.

  • Patient-derived xenograft (PDX) models to test the efficacy of MRPL17-targeted approaches in maintaining tumor heterogeneity.

  • Early-phase clinical trial designs incorporating MRPL17 expression as a stratification factor for patient selection.

• Mechanistic Studies:

  • Investigation of the impact of MRPL17 inhibition on mitochondrial translation, respiration, and metabolic pathways in cancer cells.

  • Analysis of changes in the cancer stem cell population and epithelial-mesenchymal transition following MRPL17 modulation .

  • Examination of the effects on tumor immune microenvironment, particularly focusing on the immune cell types showing significant correlations with MRPL17 expression .